Blood clots are one of the main causes of morbidity and of mortality of humans in the developed world. The network of blood clots consists of fibrin which is formed from its soluble precursor fibrinogen by the action of the enzyme thrombin. An array of enzymes and other substances ensure that clots normally form only when and where they are required to prevent loss of blood.
Mammalian plasma contains an enzymatic system, the fibrinolytic system, capable of dissolving blood clots. One component of the fibrinolytic system is a group of enzymes named plasminogen activators, which convert plasminogen (an inactive proenzyme form of plasmin) to the proteolytic enzyme plasmin. Plasmin then degrades the fibrin network of the clots to form soluble products. In cases where the thrombolytic capacity of the body is insufficient to remove intravascular thrombi, for example in patients suffering from thromboembolisms or post-surgical complications, it may be indispensable to use exogenously administered thrombolytic agents.
Two types of plasminogen activators (hereinafter referred to as "PAs") can be isolated from human body fluids or cells: urokinase or urokinase-type plasminogen activator (hereinafter referred to as "uPA"), a serine protease occurring e.g. in urine and kidney cells, and tissue-type plasminogen activator (hereinafter referred to as "tPA") which is produced by endothelial cells and found in a number of endocrine tissues.
Both tPA and uPA exist in two molecular forms. The single-chain (sc) or proenzyme form is specifically bound to components of the blood clot, such as fibrin, and is subsequently cleaved into the mature two-chain (tc) form by the action of plasmin. In the processed tcPA, the non-catalytic aminoterminal A-chain is connected via S-S bridges to the catalytic carboxyterminal B-chain.
Although ultimately cleaving the same peptide bond in the plasminogen molecule, tPA, scuPA and tcuPA exhibit unique properties, largely affecting the rate and the specificity of this reaction. Although being immunologically unrelated and having a different activation mechanism, both tPA and scuPA exhibit a high fibrin-affinity and therefore mainly activate fibrin-bound plasminogen. Free plasma plasminogen is only unsignificantly affected by tPA or scuPA. On the other hand, tcuPA, due to its lack of clot-specificity, activates both fibrin-bound and plasma plasminogen to the same extent, resulting in a systemic activation of the fibrinolytic system. The catalytic rate constant of tcuPA, however, is significantly higher than that of tPA.
Recently, a large number of hybrid PAs have been constructed by genetic engineering techniques. In these hybrids, sequences coding for parts of the tPA and parts of the uPA molecule were recombined into single chimeric molecules.
Simple hybrids are composed of the non-catalytic A-chain of one PA linked to the catalytic B-chain of the other PA (e.g. European Patent Applications Nos. 155 387 and 277 313). More complex hybrids have been constructed by recombining DNA sequences coding for discrete domains of the A-chain of tPA (which contains a "finger", a "growth factor" and two "kringle" domains) or uPA (which contains a "growth factor" and one "kringle" domain) with sequences encoding the B-chain of tPA or uPA (e.g. European Patent Applications Nos. 231 883 and 277 313 or PCT-Application No. 88/5822). "Polykringle" PAs combining in their A-chain multiple kringle structures of tPA and uPA have been described in European Patent Application No. 213 794.
The therapeutic value of tPA, uPA (especially scuPA with its high fibrin-specificity) and some of the hybrid PAs is of utmost importance in the treatment of blood clotting disorders such as thrombosis. However, often the thrombolytic action is not fast enough resulting in for example cardiac arrest in the case of myocardial infarction or incomplete leading to very rapid reocclusion of the opened vessel.
The simultaneous administration of tPA and scuPA or tPA and tcuPA as has been described in European Patent Application No. 223 192 results in a synergistic effect of the two components, i.e. a higher thrombolytic activity in vivo. Significant synergism between tPA and scuPA as well as between tPA and tcuPA has also been demonstrated in vivo by Collen and coworkers in a quantitative animal model system of thrombolysis (rabbit jugular vein with .sup.125 J-labelled fibrin clot; e.g. Collen et al. (1986) Circulation 74, 838-842; Collen et al. (1987) Thrombosis and Haemostasis 58, 943-946) and in human patients suffering from acute myocardial infarction (Collen and Van de Werf (1987) Amer. J. of Cardiology 60, 431-434; Collen (1988) Circulation 77, 731-735).
In vitro data were raised by other authors (Gurewich and Pannell (1986) Thrombosis Research 44, 217-228; Pannell et al. (1988) J. Clin. Invest. 81, 853-859) indicating that small amounts of tPA potentiate clot lysis by scuPA by attenuating the lag phase which is characteristic of scuPA and cause a much earlier transition to the rapid phase of lysis.
Because of their different mechanisms of plasminogen activation a potential synergistic or complementary mode of action between tPA and scuPA would be of significant clinical value since it would allow a reduction of the required total amount of these expensive drugs and, provided the toxic side-effects (such as allergic reactions, anaphylactic shock, intracranial hemorrhages or pyrogenic effects) would not be additive, the systemic fibrinolytic activation could be eliminated. Although a reduction of the total concentration of the PAs administered can be achieved while preserving an equivalent thrombolytic activity as described in the references cited, the applied doses, especially the one for uPA, are still too high to reliably exclude the possibility of negative side effects such as bleeding complications.
There is clearly a clinical need to overcome the problem of side effects arising in conventional thrombosis therapy. Superior fibrinolytic agents are required which are effective at relatively low doses thus minimizing possible side effects as well as saving expenses in therapy.